1. Field of the Invention
The invention relates to genetic factors associated with sensitivity to chemotherapeutic drugs. More particularly, the invention relates to methods for identifying such factors as well as to uses for such factors. The invention specifically provides genetic suppressor elements derived from mammalian kinesin genes, and therapeutic and diagnostic uses related thereto.
2. Summary of the Related Art
A broad variety of chemotherapeutic agents are used in the treatment of human cancer. For example the textbook CANCER: Principles and Practice of Oncology, 2d Edition, (De Vita et al., eds.), J.B. Lippincott Company, Philadelphia, Pa. (1985) discloses as major antineoplastic agents the plant alkaloids vincristine, vinblastine, and vindesine; the antibiotics actinomycin-D, doxorubicin, daunorubicin, mithramycin, mitomycin C and bleomycin; the antimetabolites methotrexate, 5-fluorouracil, 5-fluorodeoxyuridine, 6-mercaptopurine, 6-thioguanine, cytosine arabinoside, 5-aza-cytidine and hydroxyurea; the alkylating agents cyclophosphamide, melphalan, busulfan, CCNU, MeCCNU, BCNU, streptozotocin, chlorambucil, bis-diaminedichloro-platinum, azetidinylbenzoquinone; and the miscellaneous agents dacarbazine, mAMSA and mitoxantrone.
These and other chemotherapeutic agents such as etoposide and amsacrine have proven to be very useful in the treatment of cancer. Unfortunately, some tumor cells become resistant to specific chemotherapeutic agents, in some instances even to multiple chemotherapeutic agents. Such drug resistance or multiple drug resistance can theoretically arise from either the presence of genetic factors that confer resistance to the drugs, or from the absence of genetic factors that confer sensitivity to the drugs. The former type of factors have been identified, and include the multiple drug resistance gene mdr-1 (see Chen et al., Cell 47: 381-389). However, the latter type of factor remains largely unknown, perhaps in part because the absence of such factors would tend to be a recessive trait.
Identification of genes associated with sensitivity to chemotherapeutic agents is desirable, because the discovery of such genes can lead to both diagnostic and therapeutic approaches for cancer cells and for drug resistant cancer cells, as well as to improvements in gene therapy and rational drug design. Recently, some developments have been made in the difficult area of isolating recessive genetic elements, including those involved in cytotoxic drug sensitivity. Roninson et al., U.S. Pat. No. 5,217,889 (issued Jun. 8, 1993) teach a generalized method for obtaining genetic suppressor elements (GSEs), which are dominant negative factors that confer the recessive-type phenotype for the gene to which the particular GSE corresponds. (See also Holzmayer et al., 1992, Nucleic Acids Res. 20: 711-717). Gudkov et al., 1993, Proc. Natl. Acad. Sci. USA 90: 3231-3235 teach isolation of GSEs from topoisomerase II cDNA that induce resistance to topoisomerase II-interactive drugs. Co-pending U.S. patent application Ser. No. 08/033,986, filed Mar. 3, 1993, discloses the discovery by the present inventors of a novel and unexpected result of experiments performed to identify GSEs isolated from RNA of cells resistant to the anticancer DNA damaging agent, etoposide. This reference discloses that a GSE encoding an antisense RNA homologous to a portion of a mouse kinesin heavy chain gene has the capacity to confer etoposide resistance to cells expressing the GSE. The experiments described in this reference also demonstrate that under-expression of the particular kinesin heavy chain gene disclosed therein was associated with naturally-occurring etoposide resistance in cultures of drug-selected human adenocarcinoma cells. These results were particularly unexpected because the role of kinesin genes in etoposide resistance was unknown in the art prior to the instant inventors"" discoveries.
The kinesins comprise a family of motor proteins involved in intracellular movement of vesicles or macromolecules along microtubules in eukaryotic cells (see Vale, 1987, Ann. Rev. Cell Biol. 3: 347-378; and Endow, 1991, Trends Biochem. Sci. 16: 221-225 for reviews). Among the family of kinesin genes are encoded kinesin light chains and kinesin heavy chains that assemble to form mature kinesin. A number of kinesin genes have been isolated in the prior art.
Gauger and Goldstein, 1993, J. Biol. Chem. 268: 13657-13666 disclose cloning and sequencing of a Drosophila kinesin light chain gene.
Navone et al., 1992, J. Cell. Biol. 117: 1263-1275 disclose cloning and sequencing of a human kinesin heavy chain gene.
Kato, 1991, J. Neurosci. 2: 704-711 disclose cloning and sequencing of a mouse kinesin heavy chain gene.
Cyr et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10114-10118 disclose cloning and sequencing of a rat kinesin light chain gene.
McDonald and Goldstein, 1990, Cell 61: 991-1000 disclose isolation of a Drosophila kinesin heavy chain gene.
Kosik et al., 1990, J. Biol. Chem. 265: 3278-3283 disclose isolation of a squid kinesin heavy chain gene.
The present inventors have demonstrated that a heretofore unexpected gene, a kinesin heavy chain gene, is involved in cellular sensitivity to the anticancer drug etoposide, and that down-regulation of functional expression of this kinesin heavy chain gene is associated with resistance to this drug. Further experiments, disclosed herein, have suggested that the role of kinesin genes in chemotherapeutic drug resistance may not be limited to this single member of the kinesin gene family. These results further underscore the power of the GSE technology developed by these inventors to elucidate unexpected mechanisms of drug resistance in cancer cells, thereby providing the opportunity and the means for overcoming drug resistance in cancer patients. Reagents and methods directed towards such goals are provided in this disclosure.
The invention provides genetic suppressor elements (GSEs) that are random fragments derived from genes associated with sensitivity to chemotherapeutic drugs, and that confer resistance to chemotherapeutic drugs and DNA damaging agents upon cells expressing such GSEs. The invention specifically provides GSEs derived from cDNA and genomic DNA encoding kinesin genes. Diagnostic assays useful in determining appropriate candidate cancer patients bearing tumors likely to be successfully reduced or eliminated by administration of particular anticancer treatment modalities, including chemotherapeutic drugs and other DNA damaging agents, are provided by the invention, on the basis of levels of kinesin gene expression in the tumor cells borne by such cancer patients. In vitro drug screening and rational drug design methods are also within the scope of the instant disclosure.
The invention is based in part on the discoveries disclosed in co-pending U.S. patent application Ser. No. 08/033,086, filed Mar. 3, 1993 and incorporated by reference, providing a method for identifying and isolating GSEs that confer resistance to any chemotherapeutic drug for which resistance is possible. Particularly provided herein are methods for identifying GSEs derived from any kinesin gene, said GSEs being capable of conferring resistance to DNA damaging agents on cells expressing the GSEs. This method utilizes chemotherapeutic drug selection of cells that harbor clones from a random fragment expression library derived from kinesin-specific cDNA, and subsequent rescue of library inserts from drug-resistant cells. In a second aspect, the invention provides GSEs comprising oligonucleotides and/or peptides derived from kinesin genes that function as GSEs in vivo and confer on cells expressing said GSEs resistance to DNA damaging agents, including certain chemotherapeutic drugs. In a third aspect, the invention provides a method for obtaining GSEs having optimized suppressor activity for a kinesin gene associated with sensitivity to a chemotherapeutic drug. This method utilizes chemotherapeutic drug selection of cells that harbor clones from a random fragment expression library derived from DNA of a kinesin gene associated with sensitivity to that chemotherapeutic drug, and subsequent rescue of the library inserts from drug resistant cells. Particularly and preferably provided are such optimized GSEs derived from a mouse or human kinesin gene. In a fourth aspect, the invention provides synthetic peptides and oligonucleotides that confer upon cells resistance to DNA damaging agents, including certain chemotherapeutic drugs. These synthetic peptides and oligonucleotides are designed based upon the sequence of a drug-resistance conferring GSE derived from a mouse or human kinesin gene according to the invention.
In a fifth aspect, the invention provides a diagnostic assay for tumor cells that are resistant to one or more therapeutic DNA damaging agents and, at the same time, sensitive to therapeutic anti-microtubular agents, due to the absence of expression or under-expression of a kinesin gene. This diagnostic assay comprises quantitating the level of expression of any particular kinesin gene product in a particular tumor cell sample to be tested, and comparing the expression levels so obtained with a standardized set of cell lines expressing varying amounts of kinesin gene mRNA and/or protein and having different degrees of resistance to chemotherapeutic drugs and DNA damaging agents associated with their levels of kinesin gene expression. In preferred embodiments, such a standardized set of cell lines is matched by tissue type with the tissue type of the tumor cells to be evaluated.
In a sixth aspect, the invention provides methods for determining the appropriateness of candidates for particular cancer chemotherapeutic treatment modalities. In one preferred embodiment, the invention provides a means for determining whether a cancer patient is an appropriate candidate for treatment with DNA damaging chemotherapeutic drugs or other DNA damaging agents such as radiation, the method determining whether a kinesin gene, such as the kinesin heavy chain gene disclosed herein and in co-pending U.S. patent application Ser. No. 08/033,086, is over-expressed or under-expressed in tumor cells borne by a cancer patient, relative to a standardized set of cell lines as disclosed herein. Using this method, appropriate candidates for treatment with DNA damaging agents, including certain chemotherapeutic drugs, will be those patients whose tumor cells over-express the kinesin gene. In another embodiment, the invention provides a means for determining whether a cancer patient is an appropriate candidate for treatment with anti-microtubular chemotherapeutic drugs. Using this aspect of the method, appropriate candidates for anti-microtubular agent treatment will be those patients whose tumor cells under-express the kinesin gene compared with expression levels in a standardized set of cell lines. In a particularly useful embodiment of this aspect of the invention, potential candidate cancer patients for treatment with anti-microtubular anticancer agents will have failed or proven resistant to a course of cancer chemotherapy using DNA damaging agents.
In a seventh aspect, the invention provides a starting point for the rational design of pharmaceutical products that are useful against tumor cells that are resistant to chemotherapeutic drugs. By examining the structure, function, localization and pattern of expression of kinesin genes associated with resistance to DNA damaging agents and sensitivity to anti-microtubular chemotherapeutic drugs, strategies can be developed for creating pharmaceutical products that will overcome drug resistance in tumor cells in which such kinesin genes are either over-expressed or under-expressed.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.